Human DEAD-box helicase DDX3 has been linked to the development of many diseases such as cancers, HIV/HCV infections, and inflammation; inhibition of DDX3 may therefore produce therapeutic benefits for the treatment of these diseases. Recent study showed that a DDX3 inhibitor protects neurons in primary cortical cultures from the combined neurotoxicity of HIV-Tat and cocaine. Inhibition of DDX3 also suppresses HIV-Tat-induced microglial activation and the associated secretion of proinflammatory cytokines, both of which are responsible for neurotoxicity and the induction of neuroinflammation. As compelling evidence suggests that neuroinflammation plays an important role in developing neurodegenerative disorders including HIV-associated neurocognitive disorder (HAND) as well as age-related neurodegenerative diseases, we hypothesize that a DDX3 inhibitor may also produce therapeutic effects for the treatment of HAND. However, currently known DDX3 inhibitors have only moderate potency and poor pharmacokinetics. Therefore, we propose to design and develop more potent DDX3 inhibitors with drug-like pharmacokinetic profiles. We hypothesize that such DDX3 inhibitors may be used as chemical probes to study the interactions of DDX3 with microglia in conjunction with HIV infection. Such inhibitors may help to clarify the molecular mechanism of how DDX3 regulates neuroinflammation and help to establish DDX3 as a potential target for the treatment of HAND. Our long-term goal is to develop DDX3 inhibitors that can be used as therapeutics for the treatment of diseases such as cancers and HAND that are linked to dysfunction of DDX3. The goal of this research is to design, synthesize, and identify potent inhibitors that interrupt the binding of either ATP or RNA, or both, to DDX3 and that have drug-like pharmacokinetic profiles. These DDX3 inhibitors can be used as chemical probes to understand the molecular mechanism of DDX3 in correlation with microglia activity regarding the pathological process of HAND, or as leads to develop new drugs against cancer or HAND. The specific aims of this proposal are to: 1) design and synthesize novel DDX3 inhibitors targeting the binding regions for either ATP or RNA, or both, using crystal structures of DDX3 (in open and close conformations), molecular docking, and chemical synthesis; 2) characterize these inhibitors for their activities toward DDX3 using an ATPase assay and a helicase unwinding assay, and validate the hits by examining their on-target inhibitory effects on DDX3-dependent cancer cell lines; 3) examine the DDX3 inhibitors for their relative efficacy in blocking Tat-and-cocaine induced microglial activation; 4) evaluate lead compounds for their ADMET in vitro and pharmacokinetics using a mouse model. We believe such an effort will build the foundation to understand the role of DDX3 in the neuroinflammatory process, define a novel therapeutic target to treat HIV-associated neurocognitive disorder, and facilitate future treatment development for the diseases linked to DDX3 dysfunction.